Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy

Paco Ca´rdenas1*¤, Joana R. Xavier2,3, Julie Reveillaud4,5, Christoffer Schander1,6, Hans Tore Rapp1,6 1 Department of Biology, University of Bergen, Bergen, Norway, 2 CIBIO – Research Centre for Biodiversity and Genetic Resources, CIBIO-Azores, Biology Department, University of the Azores, Azores, Portugal, 3 CEAB – Center for Advanced Studies of Blanes (CSIC), Blanes, Spain, 4 Marine Biology Section, Biology Department, Ghent University, Ghent, Belgium, 5 CeMoFE, Center for Molecular Phylogeny and Evolution, Ghent, Belgium, 6 Centre for Geobiology, University of Bergen, Bergen, Norway

Abstract

Background: The Astrophorida (Porifera, Demospongiaep) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution.

Methodology/Principal Findings: With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deep- and shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 59 end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella).

Conclusion: The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can be explained by the banality of convergent evolution and secondary loss in spicule evolution. These processes have taken place many times, in all the major clades, for megascleres and microscleres.

Citation: Ca´rdenas P, Xavier JR, Reveillaud J, Schander C, Rapp HT (2011) Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy. PLoS ONE 6(4): e18318. doi:10.1371/journal.pone.0018318 Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada Received November 3, 2010; Accepted March 3, 2011; Published April 8, 2011 Copyright: ß 2011 Ca´rdenas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Participation of P. Ca´rdenas at the ‘Molecular Evolution Workshop’ (Woods Hole, 2009) was made possible by the Bergen Forskningsstiftelse (‘‘Mohn Grant’’, to C. Schander). Visit to the Zoological Museum in Amsterdam (P. Ca´rdenas) received support from the SYNTHESYS Project which is financed by European Community Research Infrastructure Action under the FP6 ‘‘Structuring the European Research Area’’ Program. J.R. Xavier is funded by the Science and Technology Foundation (FCT-Portugal, grant no. SFRH/BPD/62946/2009). J. Reveillaud has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under the HERMIONE project, grant agreement no. 226354. This work was supported by a grant from the ‘Norwegian Deep Sea Program’ and by the Norwegian Research Council (project number 158923/S40, to H.T. Rapp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ¤ Current address: Muse´um National d’Histoire Naturelle, De´partement Milieux et Peuplements Aquatiques, UMR ‘‘BOREA’’ 7208, Paris, France

Introduction monophyletic [1,3,4,5]. The Astrophorida is geographically and bathymetrically widely distributed around the world, and Demospongiaep Sollas, 1885 [Borchiellini et al., 2004] make up represent around 660 extant species (van Soest et al. 2010[6]; this 85% of all living sponges, and is today subdivided in 13 extant study). In tropical and parts of warm temperate waters orders. Based on molecular results, Demospongiaep are subdivided in Astrophorida species are common at quite shallow depths, while four clades: G1/Keratosap [Borchiellini et al., 2004], G2/Myx- in boreal/antiboreal and Arctic/Antarctic waters they are usually ospongiaep [Borchiellini et al., 2004], G3/Haplosclerida and G4/ deep-water species. Astrophorida species have colonized hard- as Democlavia [1,2]. The Astrophorida Sollas, 1888 are found within well as soft-bottoms from various depths. In gravely hard-bottom the Democlavia clade and represent one of the few sponge orders habitats on the outer shelf and upper slope, Astrophorida can to have been consistently and with strong support, shown to be dominate ecosystems in terms of abundance and biomass forming

PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18318 Astrophorida Phylogeny sponge grounds [7,8]. Astrophorida species display a wide array of monophyly i) of the Euastrophorida/Streptosclerophorida sub- external morphologies (massive to thin encrusting, subspherical-, orders and ii) of the Astrophorida families and genera. Our second fan-, cup- or irregularly-shaped) and external colors (Fig. 1a–d), aim was to revise the taxonomy of this order using both the and they range in size from a few millimeters to more than a meter classical rank-based nomenclature (i.e. Linnaean classification) and in diameter. There is no single morphological synapomorphy of the phylogenetic nomenclature following the PhyloCode, indepen- the Astrophorida. They are nonetheless well characterized by the dent of taxonomic rank. To be clear, names established under the simultaneous presence of star-shaped microscleres (small spicules PhyloCode are always in italics and will be identified with the symbol called ‘asters’) and tetractinal megascleres (large spicules called ‘p’ (e.g. Demospongiaep). Authors of PhyloCode names are between ‘triaenes’) (Fig. 1e–g). Star-shaped microscleres may be euasters — square brackets (e.g. Demospongiaep Sollas, 1885 [Borchiellini et al., asters in which the rays radiate from a central point (e.g. oxyasters, 2004]). Finally, our third aim was to investigate the evolution of strongylasters, spherasters, sterrasters) or streptasters — asters in Astrophorida megascleres and microscleres in order to evaluate which the rays proceed from an axis that can be straight the importance of homoplastic spicule characters in this order. (amphiasters) or spiral (e.g. spirasters, metasters, plesiasters). According to the latest major revision of the Astrophorida [9], Materials and Methods five families are included: Ancorinidae Schmidt, 1870, Calthro- pellidae Lendenfeld, 1907, Geodiidae Gray, 1867, Pachastrellidae Ethics statement Carter, 1875, and Thrombidae, Sollas, 1888. Thirty-eight genera This study has been approved by the University of Bergen and two subgenera are currently distributed in those families. In through the acceptance of a Ph.D. project proposal. an effort to incorporate some lithistids in the Astrophorida, the sub-orders Euastrophorida Reid, 1963 (Astrophorida with eua- Sponge sampling sters) and Streptosclerophorida Dendy, 1924 (Astrophorida/ Most of our collecting was done in the Northeast Atlantic. lithistids with streptasters) were erected [10,11], but in spite of Sampling in the Korsfjord (60u109N, 05u109E), Langenuen molecular evidence confirming their incorporation within the (59u539N, 05u319E) and the Hjeltefjord (60u249N, 05u059E) Astrophorida [5,12,13], lithistids have been kept apart in the (Western Norway, south of Bergen) were carried out using a Systema Porifera [14]. Other taxa such as the boring sponges Alectona triangular dredge and a bottom trawl between 40 and 500 meters and Neamphius have also been suggested to be derived Astrophor- (between the years 2005 and 2009). Southern Norway samples ida species, based on morphological [15], molecular [16] and (58u139N, 08u359E) were dredged during the BIOSKAG 2006 larval data [17,18], but they are still considered to belong to the cruise. Northern Norway samples were collected during the order Hadromerida in the Systema Porifera [19]. Polarstern ARK-XXII/1a 2007 cruise with large boxcores and the The Astrophorida is an order with one of the most diverse Jago manned-submersible. Localities sampled were Sotbakken spicule repertoire among the Demospongiaep. For example, Geodia (70u459N, 18u409E), Røst reef (67u309N, 9u249E) and Trænadjupet barretti (Geodiidae, Astrophorida) has up to ten different spicule (66u589N, 11u79E). Greenland Sea samples were collected on the types while Halichondria panicea (Halichondriidae, Halichondrida) ‘‘The Schultz Massive’’ seamount (73u479N, 07u409E) during the has only one. This spicule diversity within the Astrophorida is ideal BIODEEP 2007 and H2DEEP 2008 cruises using the ROV to trace spicule evolution and thereby evaluate the importance of Bathysaurus XL. Samples from Bocas del Toro (9u209N, 82u159E, homoplasy in this group. Homoplasy (convergent evolution and Panama, Atlantic), Berlengas Islands (39u249N, 09u309W, Portu- secondary loss) has always been acknowledged by sponge gal) and the Azores Islands were collected by snorkeling/diving. taxonomists and phylogeneticists but few studies have been able The Gorringe Bank (36u319N, 11u349W) specimens were collected to show to what extent these evolutionary processes occur in by diving during Luso Expedic¸a˜o 2006 [25]. Samples from deep- sponges, due to the paucity of spicule types and morphological water coral reefs off Cape Santa Maria di Leuca (Ionian Sea, characters. Secondary loss has been particularly difficult to reveal Apulian Plateau, 39u339N, 18u269E) were collected with the ROV in morphological studies and molecular studies of species with too Victor and an Usnel core during the ‘Ifremer MEDECO 2007’ few spicule types. Meanwhile, the paraphyly and polyphyly of cruise. Samples of the seamounts Southern of the Azores were many sponge orders in Demospongiaep and Calcarea (e.g. Haplo- collected in the course of the campaigns EMEPC-G3-2007/2008 sclerida, Halichondrida, Clathrinida, Murrayonida) in molecular of the Task Group for the Extension of the Continental Shelf phylogenetic studies clearly suggest that the evolution of spicules (EMEPC, Portugal) employing the ROV Luso. Other samples were may be more intricate than currently thought [3,4,20,21,22,23]. kindly provided by different institutions and scientists (cf. To date, the most complete molecular phylogenetic study Acknowledgments). Hologenophores — a sample or preparation focusing on the Astrophorida is based on ten species belonging to of the same individual organism as the study organism [26] — six families, including two species of lithistids [24]. Other were preserved in 95% ethanol and stored at room temperature at Demospongiaep molecular phylogenies include only three to six the Bergen Museum. Species, voucher numbers, Genbank species of Astrophorida [1,4]. With over 660 species of accession numbers and collecting localities are given in Table S1. Astrophorida described worldwide [6], needless to say that Outgroups belong to the Spirophorida since all previous p phylogenetic relationships within this order are for the most part Demospongiae molecular phylogenetic studies place them in a unknown and hypotheses based on morphology largely untested. strongly supported sister-order relationship with the Astrophorida And, since Astrophorida families might not be monophyletic [24], [1,4,5,21,27] (see also the comprehensive COI, 18S and 28S p any Astrophorida phylogenetic study needs to have the broadest phylogenetic Demospongiae trees on the Sponge Genetree Server, th sampling as possible, from the five Astrophorida families as well as www.spongegenetrees.org/, accessed on the 15 of October from putative Astrophorida (lithistids, Alectona, Neamphius). With a 2010). sampling of 153 specimens (9 families, 89 species) covering the deep- and shallow-waters of the Atlantic, Pacific, Indian, and Taxonomy Southern Ocean, the overall aim of this work was to present the Specimens collected were identified to the genus and species first comprehensive molecular phylogeny of the Astrophorida. level by P. Ca´rdenas, H. T. Rapp and J. R. Xavier. Identifications More specifically, the first aim of this study was to test the of specimens donated by other institutions were also checked.

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Figure 1. Presentation of the Astrophorida morphology. (a–d) A few Astrophorida species. (a): Geodia phlegraei (Geodiidae) collected in the Denmark Strait. Uniporal oscules are on the top surface. (b): Cross-section of a Stelletta raphidiophora (Ancorinidae) collected on the ‘Schultz Massive’ seamount (Greenland Sea) (ZMBN 85223). The grayish thick cortex is clearly visible. Specimen is 13 cm in diameter. (c): Calthropella geodioides (Calthropellidae) collected South of the Azores (ZMAPOR 21659). (d): Thenea valdiviae (Pachastrellidae) collected on the Norwegian coast. (e): Characteristic Astrophorida microscleres. ox – oxyaster of Geodia papyracea (diameter: 23 mm); st – sterraster of Geodia barretti (diameter: 80 mm); as – aspidaster of Erylus expletus (length: 330 mm); mi – microrhabd of Pachymatisma normani (length: 20 mm); pl – plesiaster of Poecillastra compressa (diameter: 37 mm); sa – sanidaster of Stryphnus raratriaenus; am – amphiaster of Characella pachastrelloides (length: 18 mm); sp – spiraster of Thenea levis (length: 23 mm). (f): cross-section of the cortex of Geodia barretti showing the skeleton organization. ec – ectocortex made of a thin layer of strongylaster and microxeas. en – endocortex made of a thick layer of sterrasters. ch – choanosome. tr – triaene supporting the cortex. Scale: 1 mm. (g): Characteristic Astrophorida megascleres. cal – calthrop of Pachastrella sp. from Norway (actine length: 100 mm); tr – long-shafted triaene of Stelletta sp. from Panama (rhabdome length: 850 mm); dicho – dichotriaene of Characella pachastrelloides (rhabdome length: 500 mm); ana – cladome of anatriaene of Geodia tumulosa from Panama (clad length: 24 mm); disco – discotriaene of Discodermia polymorpha (disc diameter: 180 mm) (photo: A. Pisera); phyllo – phyllotriaene of Theonella sp. (cladome: 730 mm) (photo: A. Pisera). doi:10.1371/journal.pone.0018318.g001

Astrophorida vouchers from previous studies [4,24,28,29,30] were Because Thrombus abyssi can have variable spicule morphologies re-examined by us or by others [31,32] and in some cases, given [40], it is important to note that our specimens have amphiasters new identifications (Table S2). Some of the voucher specimens and trichotriaenes with an extension of the rhabdome. sequenced have been morphologically described previously: Pachymatisma species [33] and all specimens collected in Panama DNA extraction, amplification and sequencing [34]. The Norwegian Pachastrellidae specimens will be described Two independent genes were used for this study: the Folmer and reviewed in a separate paper. fragment of the mitochondrial cytochrome c oxidase subunit 1 Isops and Sidonops are synonyms of Geodia [35]; Isops and Sidonops (COI) and the 59 end terminal part of the nuclear 28S rRNA gene. species of this study were therefore all transferred to Geodia. Geodia These have previously been shown to give robust and congruent neptuni Sollas, 1886 has been synonymized with Geodia vosmaeri results for Geodiidae relationships [35]. DNA extraction from Sollas, 1886 [36]. Erylus euastrum has been transferred to the genus choanosome samples was performed using the Tissue Genomic Penares, owing to molecular and morphological results [35]. The DNA extraction kit (Viogene, Sunnyvale, CA, U.S.A.) in accor- lithistid Exsuperantia sp. corresponds to Racodiscula clava sensu dance with the manufacturer’s instructions. A single centrifugation Topsent, 1892 from the Azores [37] which had been re-identified step was added just before pipeting the mixture into the columns in as Rimella sp. [38], later found to be a preoccupied genus [39]. order to remove the spicules. For some species (Pachastrella sp. and

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Stryphnus raratriaenus), polymerase chain reactions (PCR) worked only Astrophorida data matrix was distributed among hundreds of when the DNA was extracted following a standard chloroform computers, where the analyses were then conducted asynchro- protocol extraction. The 59 end region of COI (659 bp.) was nously in parallel. 100 ML search replicates were run for each amplified using LCO1490 and HCO2198 [41] (5 min/94uC; 5 dataset. Each replicate was run with a random starting topology cycles [30 s/94uC, 1 min30 s/45uC, 1 min/72uC]; 30–35 cycles and for 5,000,000 generations. Lscores of the 100 best trees from [30 s/94uC, 1 min30 s/50uC, 1 min/72uC]; 7 min/72uC). each replicate were re-estimated in PAUP* 4.0b10 [55] and trees C19ASTR (59–ACC CGC TGA ACT TAA GCA T–39) [35] and were compared using the Symmetric Difference (Robinson-Foulds) the D2 (59–TCC GTG TTT CAA GAC GGG–39) [42] reverse tree distance metric, essentially to make sure the best trees universal primer were used to amplify a 768–832 bp. region of 28S collected had similar topologies. 2,000 bootstrap replicates were comprising part of the C1 domain, and the total of the D1, C2 and conducted for each of these four datasets. D2 domains [5] (1 cycle [4 min/95uC, 2 min/59–60uC, 2 min/ To investigate spicule evolution, we reconstructed the micro- 72uC]; 35 cycles [1 min/94uC, 45 s/59uC, 1 min/72uC]; 7 min/ scleres and megascleres states at ancestral nodes on the molecular 72uC). In some cases, C19ASTR did not work and we used an tree using likelihood reconstruction methods under the Mk1 model intermediate primer instead: Ep1a’ (59–GGC AGA GGC GGR [56], with the help of Mesquite 2.74 [57] and a morphological TGC ACC–39) [5]. Sequences were then shorter, ca 690 pb (1 cycle matrix with 13 characters combined from our observations and [4 min/95uC, 2 min/59uC, 2 min/72uC]; 35 cycles [1 min/94uC, from species descriptions in the literature (Table S3). 45 s/59uC, 1 min/72uC]; 7 min/72uC). PCR products were Astrophorida species can be found at various depths. To purified using the ExoSAP-ITH kit (USB Europe, Staufen, investigate a possible relationship between depth, evolution of Germany) or gel purified using a Gel-MTM Gel Extraction System spicules and/or phylogeny, we have color-coded shallow and (Viogene). Cycle sequencing was performed using a dye-labeled deep-water species (.100 m) in the character states reconstruc- dideoxy terminator (Big DyeH Terminator v3.1, Applied Biosys- tions. Shallow submerged cave environments are prone to harbor tems, Foster city, CA, U.S.A.). Products were analyzed using an ABI deep-water sponge species [58,59], so specimens collected in Prism 3700 DNA Analyzer (Applied Biosystems). The Astrophorida shallow Mediterranean caves were considered as deep-water origin of the sequences was checked by BLAST searches (http:// species if records outside caves were in deep-water: this concerns blast.ncbi.nlm.nih.gov). Penares euastrum, Calthropella pathologica, Discodermia polymorpha and Neophrissospongia nolitangere. Stelletta lactea and Penares helleri were the Sequence alignments and phylogenetic analyses only species to appear in both shallow and deep waters. The COI data matrix includes 118 sequences (with outgroups) of which 86 are new. 245/660 characters are parsimony Phylogenetic classification of the Astrophorida informative. The 28S data matrix includes 108 sequences of Following our effort to revise sponge classification as we which 80 are new and 9 are lengthened since Ca´rdenas et al. [35]. construct new molecular phylogenies [35], we followed the 381/864 characters are parsimony informative. COI sequences principles of phylogenetic nomenclature under the rules of the were manually aligned in Se-Al v2.0a11 [43]. 28S sequences were PhyloCode v.4c (http://www.ohiou.edu/PhyloCode/) to build a first automatically aligned using MAFFT v.6.705 [44] with default phylogenetic classification based on our results. Phylogenetic parameters, implemented in SeaView v.4.1 [45]. Four insertion- nomenclature provides the opportunity to propose taxonomical deletion regions (4–20 bp long) in the D2 domain were ambiguous changes while waiting for independent evidence to confirm them, to align and regional realignments using the MAFFT’s ENSI and before implementing those changes to the more widely used strategy were computed on these four regions. The alignment was rank-based Linnaean classification. This is particularly important subsequently improved visually using Se-Al. to reduce the phylogeny/classification gap. It is also very useful for Altogether, maximum likelihood (ML) analyses were conducted intra-genera relationships (e.g. in Geodia) where the rank-based on four datasets: COI, COI amino-acids, 28S and 28S+COI. 28S classifications are insufficient to name and describe all the clades (D1-D2) and COI have been shown to evolve at similar rates [35], present [35]. We named clades that have a bootstrap higher than so the two datasets were concatenated in a single matrix 70 in the 28S+COI analysis. For the use and establishment of containing a total of 148 Astrophorida specimens (29 genera, 2 clade names, including species names, we will follow Ca´rdenas sub-genera, 89 species) and 1,527 characters, of which 811 are et al. [35]. constant, 110 are parsimony uninformative and 606 parsimony informative. For some species we had both markers, but in Results different specimens from the same region (e.g. Stelletta normani from Western Norway, Geodia megastrella from the Hebrides Islands, The best tree resulting from the COI amino-acids analyses is Pachastrella ovisternata from the NEA). The sequences of these poorly resolved with very few supported clades (Fig. S1). The best specimens were concatenated in the final matrix. Overall, we had trees from the COI analyses (Fig. S2) and the 28S analyses (Fig. a sequence for both genes for 67 specimens and 59 species of S3) are well resolved and congruent except for a few deep poorly- Astrophorida. ModelTest 3.7 [46] and ProtTest 2.4 [47] were used supported nodes. The main topology differences between the COI to find the most appropriate models of evolution respectively for and 28S trees are: i) Alectona clusters with the Spirophorida the nucleotide datasets and the amino-acid dataset. For COI, COI outgroups (28S) or with the rest of the Astrophorida (COI); ii) amino-acids, 28S and COI+28S, the models were respectively Thenea and Poecillastra+Vulcanella form a monophyletic group (28S) (according the Akaike Information Criterion): HKY+I+G, me- or not (COI); iii) Geodinaep Sollas, 1888 [Ca´rdenas et al., 2010] tREV+G, TrN+I+G and GTR+I+G. For ML runs and bootstrap cluster either with the Erylinaep Sollas, 1888 [Ca´rdenas et al., 2010] analyses we used GARLI v.0.96 [48] and Grid computing [49] (COI) or with some Ancorinidae (28S). through The Lattice Project [50], which includes clusters and The best tree from the 28S+COI analyses (Fig. 2) is fairly close to desktops in one encompassing system [51]. A Grid service for the COI tree except for the poorly-supported positions of GARLI was developed using a special programming library and Pachastrella, Poecillastra and Vulcanella (Vulcanella). From now on, we associated tools [52]. Following the model of Cummings et al. will present the results of the best tree obtained with the 28S+COI [53], who used an earlier Grid computing system [54], the dataset (Fig. 2), unless significant topology differences were observed

PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18318 Astrophorida Phylogeny in the analyses of the other datasets. Parameters estimated by Neamphius huxleyi (Alectonidae). The Ancorinidae s.s. included GARLI for the best 28S+COI tree were (lnL = 219335.557146; Asteropus, Stryphnus, Ancorina and some Stelletta (henceforth called A = 0.191611; C 0.247736; G = 0.290797; T = 0.269856; R-ma- Stelletta sensu stricto). Stryphnus and Stelletta s.s. appeared paraphyletic, trix = (1.137933 3.456486 1.476993 0.844493 4.787326); the first one because of the placement of Asteropus sp., the second pinv = 0.367474; a = 0.557592). Out of the 100 best trees (each because of Ancorina sp.. Dercitus bucklandi (Pachastrellidae) was found obtained from a different ML replicate), the first 66 trees basal to the Stryphnus+Asteropus clade. As detailed above, a few (19335.56,PAUP* lnL,19336.10) had only minor topology 28S+COI trees (with lower likelihoods) and the 28S analyses differences, essentially within the Geodinaep and the Erylinaep. The suggested that the Ancorinidae s.s. was sister-group to Geodinaep. best tree presented and discussed here is the one with the highest score (2lnL = 19335.56); it is also representative of more than half Pachastrellidae and lithistids of the trees found. The Pachastrellidae appeared as a polyphyletic group distrib- uted in four clades: clade 1) Characella pachastrelloides, clade 2) Geodiidae, Calthropellidae and Ancorinidae Pachastrella+Poecillastra amygdaloides+Triptolemma intextum, clade 3) Astrophorida (including lithistids, Alectona and Neamphius) was Poecillastra compressa+Vulcanella(Vulcanella) and clade 4) Thenea/ monophyletic in all analyses except for the 28S analyses, were Vulcanella(Annulastrella). As a result, Thenea and Pachastrella were Alectona was within the Spirophorida outgroups. Out of the 100 monophyletic while Poecillastra and Vulcanella were polyphyletic. C. best trees retrieved from the 28S+COI analyses, the first 76 trees pachastrelloides is grouping next to the lithistids. Clade 2 was found suggested identical topologies concerning the relationships be- to be sister group to the Geodiidaep clade but this was very poorly tween the Geodiidae, Calthropellidae and Ancorinidae. The supported (boostrap,50). Clade 2 moved closer to the Erylinaep Geodiidae and the Ancorinidae were not monophyletic, while and Calthropella in the COI and 28S analyses. Clade 3 and 4, both the Calthropellidae was monophyletic (but with only one genus very well-supported, appeared closer to the base of the sampled: Calthropella). Some Ancorinidae genera were distributed Astrophorida clade, but the nodes were moderately to poorly within the Geodiidae while the rest clustered in the Ancorinidae supported (bootstraps of 68 and 53). In the 28S analyses, Clade 3 sensu stricto. Furthermore, some of the Ancorinidae genera and 4 form a poorly-supported monophyletic clade. In the COI appeared polyphyletic: i) within Geodinaep (Ecionemia and Rhabdas- analyses, Clade 3 is sister-group to the Geodiidaep, the branch is trella), or ii) distributed between Geodinaep and Ancorinidaep (Stelletta). very short and poorly-supported. Melophlus sp., another Ancorinidae, clustered with Caminus vulcani The lithistids were here limited to three families two of which in the Erylinaep. (Corallistidae and Phymaraphiniidae) were only represented by a Geodiidaep Gray, 1867 [Ca´rdenas et al., 2010] is poorly single species. N. nolitangere and Exsuperantia sp. were found close to supported, but retrieved in the COI analyses (Fig. S2) and in the C. pachastrelloides but this was poorly supported (bootstraps,50). first 76 best trees of the 28S+COI analyses (Fig. 2). The 77th best With three species sampled, the Theonellidae was found tree offers a new topology: ((Geodinaep+Ancorinidae s.s.) Erylinaep). monophyletic (bootstrap of 100). When we go from tree 76 to tree 77 we go from lnL = 219937.93 to lnL = 219939.79, a significant jump in likelihood when Thrombidae and Alectonidae compared with the lnL very slow decrease from tree 51 to tree With two species sampled, the Alectonidae was found 76. We therefore also ran constrained analyses on the 28S+COI polyphyletic. Alectona millari branched between the Thrombidae dataset (100 ML replicates) forcing the Geodinaep and Ancorinidae and the rest of the Astrophorida. In the 28S analyses, Alectona was s.s. together. The best constrained tree scored a lnL = 219339.79 placed between the Cinachyrella and Craniella outgroups. Neamphius (same as our tree number 77). An Approximately Unbiased (AU) huxleyi was sister-group to the Ancorinidae s.s. but this association test using CONSEL v.0.1j [60] showed that the best constrained was not supported (bootstrap,50). In the COI analyses, N. huxleyi and unconstrained trees were not significantly different (P- branched with the lithistids, but not far away from the value = 0.395), so both topologies are plausible according to our Ancorinidae s.s.; this position was not supported either. Thrombus molecular data. We should also note that the ((Geodinaep+Ancor- abyssi is the most basal Astrophorida, branching before A. millari. inidae s.s.) Erylinaep) topology is also retrieved in the 28S analyses (Fig. S3). Geodinaep and Erylinaep were both strongly supported Maximum likelihood reconstruction of ancestral states (bootstraps of 96). Erylus and Penares were both found polyphyletic, Mapping of the 13 characters on the molecular tree gave us 13 p p with most Erylinae internodes poorly supported. Within Geodinae , trees, each with relative probabilities for every character state for p p Depressiogeodia [Ca´rdenas et al., 2010] and Geodia Lamarck, 1815 every node in the tree. We have summarized these results for [Ca´rdenas et al., 2010] were strongly supported (boostraps of 99), megascleres (Fig. 3) and microscleres (Fig. 4) by only showing p while Cydonium Fleming, 1828 [Ca´rdenas et al., 2010] was character states with 0.65.p.0.95, and p.0.95. Numerous cases moderately supported (boostrap of 86). All species for which we of spicule convergent evolution and secondary losses are revealed. had sampled more than one specimen were found monophyletic On a total of 89 species sampled, we found 43 to be shallow and except for Geodia cydonium (the British specimens were clearly 46 to be deep-sea species. If we consider secondary losses of separated from the Mediterranean/Portuguese specimens, K2P megascleres with p.0.95, we found 9 losses in shallow-species vs. 2 distance = 0,04606), Geodia gibberosa (paraphyletic) and Penares helleri losses in deep-sea species (Fig. 3). We note there are no losses of (paraphyletic). Geodia simplicissima and Geodia barretti had identical triaenes in deep-sea species. If we consider secondary losses of COI sequences. microscleres with p.0.95, we found 14 losses in shallow-species vs. A Calthropella+Geodia intuta clade appeared as sister-group to 5 losses in deep-sea species (Fig. 4). p Erylinae . This topology was poorly supported (bootstraps of 66 and Convergent evolution can be difficult to identify since we often 72) but retrieved in all ML replicates. have low probabilities for all character states in deep ancestors. With such an uncertain ancestor separating two clades, we cannot Ancorinidae sensu stricto be sure that a spicule appearing in a clade is homologous to the The Ancorinidae s.s. have the most recent common ancestors same spicule type in the other clade, or not (e.g. microxeas, with lithistids, Characella pachastrelloides (Pachastrellidae) and amphiasters). We nonetheless notice that convergent evolution is

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Figure 2. Maximum-likelihood phylogeny of the Astrophoridap using 28S+COI partial sequences from 153 taxa (89 species). Bootstrap nodal support values .50 are given at the nodes (2,000 replicates). Species names (according to the Linnaean classification) and sampling localities are given in Table S1. Names established under the PhyloCode are in italics and identified with the symbol ‘p’. doi:10.1371/journal.pone.0018318.g002 also quite frequent and concerns nearly all types of microscleres ical data [5,35]. To understand this reallocation, it was (amphiasters, toxas, sanidasters, euasters, aspidasters, microrhabds hypothesized that Penares helleri had secondarily lost its sterrasters and possibly microxeas) and megascleres (short- and long-shafted [5]. Our study confirms this reallocation by adding two other triaenes, discotriaenes, phyllotriaenes, anatriaenes, calthrops). species of Penares. Furthermore, the latter double the frequency of Desmas may have also appeared independently three times. the secondary loss of sterrasters since our results suggest that Penares is polyphyletic, just like its counterpart Erylus. Secondary Discussion loss of sterrasters therefore happened at least twice in two different newly named clades: Penaresp (P.p euastrum, P.p helleri and P.p Astrophorida and phylogenetic classification sclerobesa) and Erylusp (E.p discophorus, E.p mamillaris, E.p deficiens, E.p A phylogenetic classification of the Astrophorida, henceforth sp., E.p granularis and E.p candidata) (Fig. 4, Fig. 5). If it happened named Astrophoridap, is presented in File S1 and summarized in twice, it could have happened more, and this is what the Figure 5. Names have been given to the well-supported clades placement of Erylusp sp. (an Erylus with no aspidasters) and other (boostraps .70). Rank-based names have also been given to clades genera of Ancorinidae within the Geodiidaep suggest: Melophlus sp., for which no names existed in the Linnaean classification. Rhabdastrella, Ecionemia, and some Stelletta would have also lost their Moreover, new definitions of families and genera were also sterrasters (Fig. 4). As in the example of Penaresp, this is fairly easy required. The revised Astrophorida Linnaean classification is to conceive since these Ancorinidae species share i) spicule presented in File S2. repertoires identical to the Geodiidaep except for the presence of Very early on, sponge taxonomists subdivided the Astrophorida sterrasters, and often ii) a similar external morphology (e.g. oscule between those that possessed streptaster and those that possessed organization). Despite these similarities, the placement of the euasters [15]: Streptosclerophorida and Euastrophorida respec- polyphyletic Rhabdastrella and Ecionemia within the Geodinaep is not tively. Chombard et al. [5] previously found the Euastrophorida straightforward. monophyletic and the Streptosclerophorida paraphyletic because Based on the possession of microrhabds in the cortex, p they had mainly sampled Geodiidae species, except for Stryphnus Chombard et al. [5] wondered if Ecionemia should be reallocated mucronatus that they had classified as a Streptosclerophorida (on the to the Erylinaep. Our analysis suggests that the three Ecionemia basis that its sanidasters were homologous to streptasters). species sampled belong to the Geodinaep, and are distributed in two However, our study suggests that both sub-orders are polyphyletic groups. The two Australian Ecionemia group with some Stelletta — (irrespective of the nature of the sanidasters of Stryphnus). thus forming the new clade Geostellettap — while Ecionemia Therefore, we propose to formally abandon the two suborders megastylifera from the Caribbean is branching at the base of Euastrophorida and Streptosclerophorida. Cydoniump. These three species of Ecionemia all share large spiny microrhabds in the cortex along with euasters. Since microrhabds Geodiidaep and reallocated Ancorinidae are absent from all the other Geodinaep of this study, the origin of Since the last molecular phylogeny of Geodiidaep [35], we these microrhabds is uncertain at this point and may represent yet lengthened the 28S sequences and increased the sampling from 24 another case of morphological spicule convergence in sponges to 38 Geodiidae species and from 24 to 62 Geodiidae specimens. (Fig. 4). Other species of Ecionemia, with small sanidaster-like We also added species from phylogenetically close families microrhabds (e.g. E. acervus, type species of the genus, E. demera, E. (Ancorinidae and Calthropellidae). Clearly, Geodiidaep is poorly walkeri), might instead be linked to sanidaster-bearing Ancorinidaep supported in our 28S+COI best tree (Fig. 2), but morphological as previously suggested [61,62,63]. In our opinion, the genus data [35] and a majority of our 28S+COI best trees support the Ecionemia should therefore be kept valid for the remaining species Erylinaep+Geodinaep grouping. This is therefore the topology we will of Ecionemia whose phylogenetic positions remain to be tested. discuss in this paper. However, as we stated earlier (cf. Results), the Based on its spicules and skeleton organization, Rhabdastrella has alternative topology Erylinaep(Geodinaep+Ancorinidae) found in a previously been suspected to be close to the Geodiidae [64] or few 28S+COI searches and 28S analyses could not be rejected on even part of the Geodiidae [65]. Biochemical data also concurs statistical grounds. The contentious Geodiidaep node should with this result: isomalabaricane triterpenes have been found in R. therefore be investigated further with additional molecular globostelletta and Geodia japonica [66,67]. Rhabdastrella species from markers. our study are distributed in three groups: 1) R. globostelletta and The Geodiidae is here redefined: it appears as a much larger Rhabdastrella sp. form a clade of uncertain position within the family than expected since it includes genera from the Calthro- Geodinaep,2)R. cordata from Australia forms a strongly supported pellidae and Ancorinidae. This is surprising for a group whose group with Geodiap pachydermata and Geodiap sp. 2, both from the monophyly and morphological synapomorphies appear quite Atlantic/Mediterranean area, and 3) R. intermedia forms a strongly clearly [35]. To understand this, we must consider the morphology supported clade with Geodiap phlegraei. Rhabdastrella species are of the unexpected groups. The Ancorinidae is partly composed of characterized by sterrospherasters in the cortex. Sterrospherasters species which have the same set of spicules as the Geodiidae except is a general ambiguous term that includes two main types of large for the presence of sterrasters (ball-shaped euasters, Fig. 1e). euasters: i) very large spherasters with smooth conical rays, filling Consequently, these Ancorinidae may have never had sterrasters the whole cortex (e.g. R. globostelletta and Rhabdastrella sp.) or ii) or they may have secondarily lost them. In the second case, these sterrasters, sometimes with incompletely fused actines (e.g. R. rowi, species should be reallocated within the Geodiidae. R. aurora, R. cordata), placed in the endocortex. These morpholog- Penares is one of these former Ancorinidae genera reallocated to ical observations coupled with our results suggest that these the Geodiidae based on morphological, molecular and biochem- sterrospherasters might actually be, in the first case, true

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Figure 3. Presence and absence of megasclere spicules mapped on the Astrophoridap 28S+COI ML tree from Figure 2. The ML reconstructions of the ancestral conditions at the nodes were estimated using Mesquite 2.74. For the readers’ convenience, species clades have been reduced to one sample (except in cases of para- or polyphyletic species). Species names in blue represent deep-water species. Species names in black represent shallow-water species. For the color-codes of the Astrophorida families sensu Systema Porifera, see Figure 2. doi:10.1371/journal.pone.0018318.g003 spherasters — they resemble the ones found in the phylogenet- their 28S D2 domain while ZMAPOR 21231 (Morocco) appeared ically close G.p phlegraei and G.p angulata — and are, in the second to have a slightly different sequence, notably without the deletion. case, true sterrasters. Rhabdastrella with true spherasters may This specimen’s morphology needs to be further investigated as therefore have secondarily lost their sterrasters (and these have G.p megastrella may represent a species complex. been replaced by the large spherasters). In light of these results we The two deep-water Geodiap species from New Caledonia expect all Rhabdastrella species to be redistributed in Geodinaep. The grouped together but this is poorly supported. The most basal genus Rhabdastrella is therefore not valid and should be synony- Geodinaep was a strongly supported clade named Synopsp grouping mized with Geodia. As a consequence of the polyphyly of G.p pachydermata, Geodiap sp. 2 and R. cordata. The surprising Rhabdastrella, the confusing spicule term ‘sterrospheraster’ should phylogenetic position of Geodia intuta with Calthropella will be be once and for all rejected, as suggested before [68]. discussed below. The positions of other Geodinaep species (e.g. G.p We should not be surprised to find Ancorinidae species with phlegraei, G.p angulata) were poorly supported and uncertain microrhabds such as Melophlus sp. grouping with Caminus vulcani (an (different positions in different trees) so we cannot discuss their Erylinaep with spherules) since it has been argued that spherules taxonomy at this point. may have evolved from microrhabds [35]. Furthermore, like the rest of the Erylinaep, Melophlus sp. has no ana/protriaenes. The Erylinaep p phylogenetic position of Melophlus sp. among the Erylinae may be Erylinaep was a very strongly supported group (boostrap of 96). further supported by biochemical data: sarasinoside M, a The monophyly of Erylus has been previously challenged by triterpenoidal saponin isolated from Melophlus sarassinorum, has morphological and molecular data [33,35]. Our results suggested strong similarities with the framework of Eryloside L, isolated in that it was a polyphyletic genus, mixed with Penares, Caminus, Erylus lendenfeldi [69]. Melophlus and Pachymatismap species. Erylus species were distributed To conclude, the reallocation of numerous Ancorinidae species in in three clades: Erylusp (‘nomen cladi conversum’ because it holds the the Geodiidae calls for new definitions for these families (File S2). type species of Erylus: E.p mamillaris), Penaresp (‘nomen cladi conversum’ because it holds the species type of Penares: P.p helleri) and Erylus1 Geodinaep (temporary name for the clade including E. aleuticus+E. expletus+E. Most of the clades found in this study are identical to those topsenti, poorly supported). If Erylus is polyphyletic, the most found previously with fewer species and a shorter 28S fragment parsimonious scenario is that flattened sterrasters ( = aspidasters) [35]. Geodiap, Cydoniump and Depressiogeodiap were still strongly have appeared independently at least three times; this is also supported groups. The Depressiogeodiap+Cydoniump clade, poorly suggested by our character reconstruction using ML methods supported in Ca´rdenas et al. [35], was better supported here (Fig. 4). Our study has not revealed the identity of Erylusp sp. (bootstrap of 77), it exclusively grouped Atlantic species. In the collected in the Gorringe Bank [25]. Erylusp sp. which has lost its following paragraphs, we will go through these clades and discuss aspidasters was part of the E.p mamillaris/discophorus complex, but new taxonomical results that have arisen due to the addition of more rapidly evolving markers are required to fully understand new species since Ca´rdenas et al. [35]. this group. The addition of Geodiap corticostylifera from Brazil confirmed that the Geodiap include species from North and South America, from Calthropellidae and Geodia intuta the Atlantic and Pacific sides. Different clades of Geodiap vosmaeri The association of calthrops and euasters essentially character- (former G.p neptuni) appeared, two from Florida, another from izes the Calthropellidae. According to some morphologists, the Belize+Bahamas suggesting i) a strong geographical structure and Calthropellidae do not really have characters of their own and that ii) the molecular markers used may be suited for future intra- should be within the Ancorinidae [70,71,72]. However, the first specific studies. Our results confirmed that Geodiap gibberosa molecular evidence suggested a sister-group relationship between represented a species complex, as previously hypothesized with the Calthropellidae and the Erylinae [5]. Although the Erylinaep(G. morphological observations [34]. We propose that G.p tumulosa intuta+Calthropellap) association was weakly supported (bootstrap of Bowerbank, 1872 (a synonym of G.p gibberosa) should be 66) it was present in all our trees obtained from the 100 ML resurrected for the mangrove specimen from Panama. Its tumulose searches. Furthermore, the external morphology of Calthropellap shape is clearly different from the barrel-shape of our reef geodioides and some basal Erylinaep species (e.g. E. expletus) is quite specimens from Belize and Mexico, more similar to the shape of similar: they are massive sub-spherical sponges with numerous the holotype of G.p gibberosa (specimen MNHN DT-608). white uniporal oscules on the top surface. We propose to reallocate Geodiap conchilega and E. megastylifera are part of Cydoniump so this the Calthropellidae to the Geodiidae by downgrading them to a clade still gathers Atlanto-Mediterranean species. The polyphyly sub-family: the Calthropellinae. Paxataxa and Corticellopsis are the of Geodiap cydonium calls for a revision of this species whose other genera of the Calthropellinae since Chelotropella has been taxonomical history is old and complex. reallocated to the Ancorinidae [73]. Sequences of Pachataxa and Geodiap megastrella is part of the Depressiogeodiap. This clade thus Corticellopsis are therefore needed to confirm the monophyly and remained a Northeast Atlantic deep-water species group. The the position of this group. inclusion of G.p megastrella in the Depressiogeodiap also confirmed a The clustering of Geodia intuta with Calthropellap was surprising, suggested morphological synapomorphy of the group: a deep but less so when reconsidering its external and spicule morphol- preoscule lacking sterrasters in its cortex [35]. It should be noted ogies. Like Erylusp and Penaresp, G. intuta is a massive sub- that the G.p megastrella ZMBN 85208 (Scotland) and ZMAPOR hemispherical sponge with a smooth cortex, it is easily compress- 21654 (Azores) both had a distinct large deletion (35 bp long) in ible, and has a rather confused skeleton organization. It was

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Figure 4. Presence and absence of microsclere spicules mapped on the Astrophoridap 28S+COI ML tree from Figure 2. The ML reconstructions of the ancestral conditions at the nodes were estimated using Mesquite 2.74. For the readers’ convenience, species clades have been reduced to one sample (except in cases of para- or polyphyletic species). Species names in blue represent deep-water species. Species names in black represent shallow-water species. For the color-codes of the Astrophorida families sensu Systema Porifera, see Figure 2. doi:10.1371/journal.pone.0018318.g004 originally described as an Isops because of its uniporal oscule and considered that the origin of the toxas being ambiguous, emphasis pores. According to our observations, the oscule actually leads to a should instead be placed on the presence of calthrops, which had branching atrium, similar to the ones found in Erylusp, Penaresp or brought Dercitus closer to the Pachastrellidae [15,81,82,83]. D. Caminus. This prompted von Lendenfeld [74] to describe it in a bucklandi as an Ancorinidaep suggests that toxas would have new genus, as Caminella loricata, before it was synonymized with originated from asters, as previously hypothesized [75]. The Geodia intuta [75]. Moreover, it has long-shafted triaenes (as in the modification of oxyasters into toxa-like spicules is actually quite Geodinaep) but no ana/pro/mesotriaenes (as in the Erylinaep). It has common in the Astrophoridap (e.g. Erylus nummulifer, Erylus expletus, spherasters in the ectocortex and globular sterrasters in the Geodia apiarium, Erylus papulifer, Rhabdastrella oxytoxa and Stelletta endocortex. Globular sterrasters are also present in many Erylinaep toxiastra). The difference between the latter and D. bucklandi, which (e.g. Caminus, Pachymatismap, E. topsenti). As for spherasters, they troubled morphologists, is that toxas in D. bucklandi have resemble the spherules found in C. vulcani (an Erylinaep)or completely lost trace of the original euaster centrum. The position Calthropellap durissima. All in all, although G. intuta shares many of D. bucklandi also shows that its sanidasters are homologous to characters with some Erylinaep (Erylusp, Penaresp, Caminus), the those of Stryphnusp (Fig. 4). Unfortunately, we did not get 28S presence of long-shafted triaenes and the absence of microrhabds sequences for D. bucklandi and the strongly supported Stryphnusp+D. suggest that it is not an Erylinaep. Therefore, we decided to bucklandi clade needs to be confirmed before resurrecting the resurrect the Geodiidae genus Caminella von Lendenfeld, 1894 to Sanidasterinae Sollas, 1888, characterized by the possession of welcome this species. On the other hand, we will wait for further sanidasters. Furthermore, Stoeba (not sampled here) having been data to confirm its phylogenetic position and name the G. synonymized with Dercitus [73], we can be confident that Stoeba intuta+Calthropellap clade. species should also be reallocated to the Ancorinidaep.

Ancorinidaep The polyphyletic Alectonidae Ancorinidae sensu stricto form a well-supported clade henceforth The Alectonidae Rosell, 1996 (Hadromerida) are excavating named Ancorinidaep. Stelletta species were distributed in three sponges recently separated from the rest of the Clionaidae Ancorinidaep clades: clade 1) (Ancorina sp.+Stelletta sp. 1)+Stelletta d’Orbigny, 1851 notably due to the possession of amphiasters or clarella, clade 2) (Stelletta normani+Stelletta raphidiophora)+Stelletta lactea microrhabds, and absence of tylostyles. Alectona are known to and clade 3) (Stelletta grubii+Stelletta carolinensis)+Stelletta dorsigera. produce a unique type of larva in the Porifera: an armored Clade 1 was poorly supported (bootstrap,50). Clade 2 clustered planktonic larva ( = hoplitomella larva) with discotriaenes [17,18]. three Northeast Atlantic species; it was very well supported by our These are then lost by the adult, which settles and bores into data (bootstrap of 98) and by the synapomorphy of trichodragmas biogenic substrata such as calcareous rocks or coral. The (raphides in bundles) (Fig. 4): it was therefore named Dragmastrap. association of triaenes and amphiasters suggest that Alectona should Clade 3 held the type species of the genus (S. grubii) so it was be placed near or within the Tetractinellidap [Borchiellini et al., named Stellettap. It should be noted that S. dorsigera does not group 2004] [17,84]. A 28S (D1-C2) phylogenetic study then showed with S. grubii in the 28S analyses (Fig. S3). The unstable position of that the Alectonidae sensu Ru¨tzler [19] is polyphyletic and that S. dorsigera may be due to the fact that the Stelletta COI sampling is Alectona millari belonged to the Tetractinellidap [16]. Our data not quite poor with respect to the Stelletta 28S sampling. The grouping only confirmed this but also suggested that the Alectonidae genera of clade 1+Dragmastrap is poorly supported or absent (28S analyses) Alectona and Neamphius belonged to the Astrophoridap. In the but we nonetheless note that all of these species have dicho- 28S+COI analyses, A. millari branched after Thrombus abyssi,an triaenes, except for Ancorina sp.. Conversely, species in the Stellettap acknowledged Astrophoridap. In the 28S analyses, Alectona appeared clade do not possess dichotriaenes. Instead, 28S analyses fully within the Spirophorida outgroups branching between Cinachyrella support a Dragmastrap+Stellettap clade (Fig. S3). and Craniella (Fig. S3), but the node between A. millari and Craniella Since Ancorina and Stryphnus share similar spicule repertoires sp. is not supported, and the branch is short. This result may be [34], notably the presence of sanidasters (Fig. 4), we were due to the fact that the Alectona 28S sequence is significantly shorter expecting them phylogenetically closer to each other than here (409 bp.: D1-C2 domains) than the others sequences from this observed. But the grouping of Ancorina sp. with two Stelletta species study. The ambiguous position of Alectona certainly deserves was poorly supported and may be due to the poor sampling of further investigation as it may represent a pivotal evolutionary step these speciose genera. between Astrophorida and Spirophorida. The close relationship between Asteropus and Stryphnus has often Having amphiasters but no triaenes, Neamphius huxleyi (the single been discussed [15,34,76,77,78,79]. Both genera have similar species of its genus) has also been suspected to be an Astrophorida spicules, except for triaenes that Asteropus would have secondarily by morphologists [15]. According to our results it may be close to lost (Fig. 3). For the first time, the synonymy of Asteropus with Characella and the lithistids. This is further supported by Stryphnus is confirmed by molecular results. Therefore, we formally biochemical data showing that N. huxleyi and Astrophorida propose that Asteropus becomes a junior synonym of Stryphnus and lithistids (Callipelta sp., Theonella mirabilis and Theonella swinhoei) name this clade Stryphnusp. share cyclic peptides and depsipeptides with cytotoxic and antiviral The presence of Dercitus bucklandi — a Pachastrellidae with effects, notably with HIV-inhibitory activity [85,86]. However, the calthrops, sanidasters and toxas — within the Ancorinidaep is once position of N. huxleyi being equivocal and poorly supported, we more supported by morphological data. Dercitus (Stoeba included) propose to temporarily consider it as incertae sedis. and Stryphnus notably share sanidasters, large spherulous cells, and Our results also have consequences for the rest of the a similar aquiferous system [70,75,80]. But other authors had Alectonidae genera. Following Borchiellini et al. [16], we advocate

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Figure 5. Phylogenetic classification of the Astrophoridap on the 28S+COI ML tree (cf. File S1 for definition of names). Species names are given according to the PhyloCode (Article 21.5). Bootstrap nodal support values of clades defined by the PhyloCode are given (2,000 replicates). For the color-codes of the Astrophorida families sensu Systema Porifera, see Figure 2. doi:10.1371/journal.pone.0018318.g005 the reallocation of Thoosa along with Alectona. Delectona might also a less reliable character, since these can be more ambiguous to join them since it shares amphiasters and toxas with Thoosa. These characterize (cf. new definitions in File S2). Due to a lack of three genera (representing ca 29 species) would group in the robustness, we propose to have Characella as incertae sedis at the Thoosidae Rosell and Uriz, 1997, here resurrected. The position moment, although we suspect that it could be allocated to a of the rest of the Alectonidae (Spiroxya, Dotona and Scolopes) is at the lithistid family in the future. moment uncertain although Spiroxya and Dotona are suspected to be According to the ICZN and our results, the Pachastrellidae phylogenetically close to each other [19]. On the Sponge Gene name should be kept for the Pachastrella+Triptolemma clade, Tree Server (www.spongegenetrees.org [87], accessed on the 15th henceforth named Pachastrellap. Until further molecular data, we of October 2010), a phylogenetic 28S (B9-B21) tree of the propose to include Poecillastra amygdaloides in this newly defined Demospongiae suggested that Spiroxya levispira should remain close Pachastrellidae (File S2), although its position was poorly to the Placospongidae and the Trachycladidae (Hadromerida). supported. P. amygdaloides has calthrops: this species and its synonym Poecillastra debilis had therefore originally been described Thrombidae as Pachastrella [90]. But P. amygdaloides was moved to Poecillastra Since Le´vi [82], the puzzling Thrombidae have been linked to because of its atypical triactinal calthrops, with a reduced fourth the Astrophorida, based on their unique amphiasters and actine, later considered to be a modified triaene [15,80]. Its sister- trichotriaenes. With the discovery of Yucatania sphaerocladoides,it group position with Pachastrellap is supported by its spicule appeared clear that Thrombus species had secondarily lost their characters which seem intermediate between the Poecillastra+Vul- triaenes [88], which confirmed that they belonged to the canella(Vulcanella) clade and Pachastrellap: i) plesiasters (most of them Tetractinellidap. Our study showed that Thrombus abyssi is alone, at are amphiaster-like) and ii) no microstrongyles. Other species (not the base of the Astrophoridap tree which suggests, as for Alectona, the sampled here) share the triactinal calthrops with P. amygdaloides: key role of this group in understanding how and when the Poecillastra nana, Poecillastra connectens and Characella capitolii.We Astrophoridap originated. propose to resurrect Nethea Sollas, 1888 (originally defined as resembling Poecillastra but with triaenes with an underdeveloped The Pachastrellidae and the lithistids rhabdome) to welcome these species. Triptolemma are cryptic The latest revision of the Pachastrellidae includes 12 genera [81] excavating species penetrating the tissue of other sponges or coral. p which share streptasters (rays proceeding from an axis that can be Many morphological characters support the Pachastrella clade straight or spiral, Fig. 1e) and do not have euasters (rays radiating claimed by Topsent [80]. Triptolemma are characterized by short- from a central point, Fig. 1e). Topsent [80] suggested that the shafted mesotriaenes of all sizes, which can be also produced by Pachastrellidae could be subdivided between those that share a some Pachastrella species (e.g. P. ovisternata). Microscleres of diverse set of streptasters (Thenea, Vulcanella, Poecillastra, some Triptolemma are streptasters (from only amphiasters to a diverse Corallistidae) and those whose streptasters are mainly restricted to set), microstrongyles and even microrhabdose streptasters [91]. amphiasters (rays radiating from both ends of a straight shaft, Fig. 1e) These last two microscleres are apomorphies shared with (Pachastrella, Characella, most Astrophorida lithistids). However, in Pachastrella. Brachiaster (not sampled here) surely belongs to this our study, none of these groups were monophyletic (Fig. 2). We clade since it also produces short-shafted mesotriaenes, micro- sampled six Pachastrellidae genera and they were distributed in five strongyles and amphiasters [92]. different clades: clade 1) Dercitus was reallocated to the Ancorinidaep Thenea, Vulcanella, and Poecillastra share a diverse set of (cf. above); clade 2) Characella appeared at the base of the Ancorinidaep streptasters [80]. Poecillastra+Vulcanella(Vulcanella) further share i) along with lithistids and Neamphius; clade 3) Poecillastra amygdaloides an oscule area surrounded by cloacal oxeas (in Poecillastra compressa +Pachastrella+Triptolemma was the sister clade of the Geodiidaep. this area has expanded over a whole side of the sponge but the Although the positions of Characella and clade 3 were poorly cloacal oxeas are still there), ii) an abundance of spiny microxeas, supported and unstable depending on the dataset (Fig. S2, S3), they iii) a reduction of the triaenes to short-shafted triaenes or calthrops were clearly separated from the other Pachastrellidae genera (even if long-shafted triaene species also exist) and iv) an absence of branching further down in the tree: clade 4) Poecillastra+Vulcanella(- pro/anatriaenes (except in Poecillastra rudiastra). In order to Vucanella) and clade 5) Thenea+Vulcanella(Annulastrella). Clearly the welcome this very well supported clade named Vulcanellidaep,we Pachastrellidae were built on a plesiomorphy (the streptasters) and created the Vulcanellidae fam. nov. (File S2). On the other hand, the family must be revised. the Thenea+Vulcanella(Annulastrella) clade was poorly supported Characella is defined by amphiasters and at least two categories of (bootstrap,50). And yet, these two genera share i) large plesiasters monaxonic spicules (microxeas, microstyles, microstrongyloxeas) and ii) absence of microxeas. For the time being, the Theneidae while Poecillastra is defined by a diverse set of streptasters Carter, 1883 is resurrected to welcome these two genera. Also, (spirasters, metasters and plesiasters) and microxeas in a single Vulcanella(Annulastrella) needs to be upgraded to genus since it was category [81]. As Characella has been occasionally difficult to clearly separated from Vulcanella(Vulcanella). The Thenea clade, here characterize with respect to Poecillastra, morphologists have named Theneap, is very well supported (boostrap of 93) and also questioned their validity [72,76,89]. Their definitions may overlap one of the few clades supported by the COI amino acid analyses and many species are found to be ‘‘intermediate’’, with characters (tree not shown). It groups species that share i) a characteristic of both genera (e.g. Poecillastra saxicola). According to our results, external morphology (massive, hispid mushroom shape, Fig. 1d), Characella was clearly separated from Poecillastra and phylogenet- with ii) a typical poral area, iii) long-shafted dichotriaenes (never ically closer to amphiaster-bearing lithistids. The definitions of calthrops), iv) an abundance of pro/anatriaenes and v) a system of Characella and Poecillastra should therefore prioritize the nature of roots to grow on muddy bottoms. Based on morphology, streptasters and consider the number of categories of microxeas as Cladothenea (not sampled here) should belong to this clade [81].

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The Theneidae and the Vulcanellidae fam. nov. may i) form a Evolution of Megascleres in the Astrophoridap (Fig. 3) poorly-supported clade (28S analyses, Fig. S3), ii) have a Astrophoridap species are well characterized by the simultaneous paraphyletic relationship (28S+COI tree, Fig. 2) or iii) be further presence of asters (microscleres) and triaenes (megascleres) apart (COI analyses, Fig. S2). All of these poorly supported (Fig. 1e–g). Therefore, the classification of this order has essentially topologies emphasize that relationships between these two families been based on variants of these two spicule types. The triaene is a remain to be investigated. synapomorphy of the Tetractinellidap so it appeared in the common As previously suggested by morphological [10,15,70,93,94,95] ancestor of Spirophorida and Astrophoridap. Since then, it has and molecular data [5,12,13], our phylogeny confirmed that some evolved in different directions giving rise to numerous descriptive p lithistids belong to the Astrophorida . The Discodermia+Theonella clade terms with respect to the cladome orientation (ortho/plagio/pro/ p named Theonellidae was strongly supported (bootstrap of 100). meso/anatriaenes), cladome branching (phyllo/disco/dicho- According to morphology and a previous 18S phylogenetic study, triaene) or the rhabdome length (long-shafted/short-shafted/ Racodiscula may also be part of the Theonellidaep [13]. We note that pseudocalthrops/calthrops). According to our data, the presence Discodermia has microxeas and microrhabds while Characella of triaenes or anatriaenes is not likely in the common ancestor of (phylogenetically close to Discodermia in our tree) has two sizes of Astrophoridap (Fig. 3). This is probably due to the presence of microxeas. The microrhabds of the Theonellidaep might therefore be Alectona and Thrombus at the base of the tree, both without triaenes. homologous to the small microxeas of Characella. We also notice Long-shafted triaenes possibly appear (p = 0.68) in the ancestor of that the microrhabds of Discodermia are similar to the ones found in the Theneidae and the rest of the Astrophoridap. Since then, they Pachastrella (e.g. Discodermia proliferans): these might also be have evolved into short-shafted triaenes or calthrops. Calthrops homologous. Exsuperantia sp. (Phymaraphiniidae) is morphologi- have appeared independently many times (Calthropellap, Pachas- cally very close to the Theonellidaep, but it has trider desmas instead trellap, Dercitus, some Vulcanella), and so have mesocalthrops and of tetraclone desmas. Exsuperantia sp. either groups with Characella mesodichotriaenes (Calthropellap, some Pachastrellap). Concerning (28S+COI and 28S dataset), or with N. huxleyi (COI analyses). In anatriaenes, our analyses (Fig. 3) suggest that they have appeared both cases, the support was low. Morphological [82] and independently many times (in Theneap, Characella, some Stelletta, molecular [13] data suggest that the Corallistidae is a sister-group Geodinaep). Discotriaenes have appeared independently in some to the Theonellidae. Because of the low supported nodes between lithistid Astrophoridap (e.g. Discodermia) and in the larvae of Alectona, our lithistids this cannot be excluded: the position of Neophrissos- although we cannot rule out the possibility that they are present in pongia nolitangere (Corallistidae) is unsure but certainly close to the other Astrophoridap larvae (never observed to date). Phyllotriaenes other lithistids. Our results also hint that desmas have appeared are only known in some lithistid families, but may have appeared independently in different Astrophorida lithistid groups (at least independently at least twice (Phymaraphiniidae and Theonella). To four times, if we would consider Brachiaster, not sampled here) conclude, most variants of triaenes are clearly the product of (Fig. 3). This would not come as a surprise since desmas have convergent evolution and thus homoplasic characters that cannot appeared independently in other sponge orders as well [96]. It be used for Astrophoridap classification. On the other hand, they should be emphasized that, in our opinion, 8 out of the 13 extant may still represent apomorphies at lower ranks. lithistid families are of Astrophorida affinities (Corallistidae, Before going further, we should clarify the term ‘secondary loss’. Isoraphiniidae, Macandrewiidae, Neopeltidae, Phymaraphiniidae, An ‘absence’ state can be optimized as a plesiomorphy (true Phymatellidae, Pleromidae, Theonellidae) representing ca 128 absence), a homoplasy (independent secondary losses which species [6]. A majority of them possess amphiaster streptasters appeared through convergent evolution) or a synapomorphy while the remaining groups have additional spirasters (Corallisti- (unique secondary loss shared by a single clade) [97]. In this last dae, Pleroma) or no asters (Macandrewiidae, Discodermia, Theonella). case, ‘absence’ states may also potentially bring phylogenetic Therefore, although Astrophoridap lithistids do not seem to form a information. Furthermore, a spicule secondary loss can be i) a natural group, we can be certain that they all radiated along with ‘true’ loss when nothing replaces the spicule lost (e.g. loss of amphiaster-bearing Astrophoridap (Characella, Pachastrella, Tripto- sterrasters) or ii) a ‘semantic’ loss by modification of a spicule into lemma, Brachiaster, and Neamphius). If they have a closest common another (e.g. sterrasters becoming aspidasters). It may not always ancestor with the Ancorinidaep, the Geodiidaep, or both, is still unclear be possible to discriminate a ‘true’ loss from a ‘semantic’ loss. For at this point. example, secondary loss of triaenes is ambiguous because some The node following that of the Vulcanellidae may be of species may have retained megascleres derived from triaenes, such importance since it supports, albeit moderately, a clade comprising as styles while others may have really lost their triaenes. We amphiaster- and euaster-bearing Astrophoridap (Fig. 4), temporarily therefore considered that when styles were present, it was a called ‘clade A’ (Fig. 2). Our study thus reveals for the first time the semantic loss, because when only oxeas remained it had a higher importance of amphiasters in Astrophoridap aster evolution, as an chance of being a true loss of triaenes. intermediate step between spirasters and euasters. The shortening Our study shows that triaenes have been secondarily lost (with of the amphiaster central shaft may represent an essential and p.0.65) independently at least four times in our sampling (e.g. preliminary stage to the appearance of euasters. Clade A includes Melophlus, Asteropus, Vulcanella (Annulastrella), Neamphius) and mor- all the Astrophoridap except for the Vulcanellidae, the Theneidae, phology suggests that it may have happened in even more Alectona and Thrombus, but since the position of the Vulcanellidae is Astrophorida taxa, not all sampled here (Thrombus, Lamellomorpha, unstable, so is the content of clade A. We thus refrain from Holoxea, Jaspis, some Stelletta, some Rhabdastrella, some Erylus, some formally naming clade A and wait for confirmation from other Geodia) [62,78,83,98]. We observe similar results for anatriaenes molecular markers. Lamellomorpha strongylata Bergquist, 1968 incertae which may have been lost eight times independently. It is also sedis (not sampled) lacks triaenes and possesses only two types of worth mentioning that anatriaenes do not seem to have been lost microscleres: spiny microstrongyles and amphiaster-like strepta- in the Erylinaep as suggested before [35]. According to our results sters. This species could therefore belong to the amphiaster/ (Fig. 3), the common ancestor of the Geodiidaep did not have euaster-bearing clade, and may be phylogenetically close to anatriaenes, they only seem to appear in the Geodinaep. Their Characella or to Pachastrellap, both of which have small ectosomal absence should therefore not be considered as a synapomorphy of monoaxial spicules. the Erylinaep [35] but as a plesiomorphy.

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Our results clearly demonstrate how common secondary loss of some lithistids and Characella (if we consider that small microxeas a megasclere is, even when this megasclere has a clear function: present in the cortex are microrhabds). In some cases, such as in providing support of the cortex, organization of the choanosome the Erylinaep, they might be derived from asters [35]. The or even defending against predators. Secondary loss of triaene is a appearance of microxeas in the ancestor of the Vulcanellidaep homoplasic character for the Astrophoridap, but it may become might also be linked to asters. In the Theneidae, plesiasters synapomorphic in more restricted clades (e.g. Vulcanella (Annulas- reduced to two actines are common: they look like microxeas and trella), Melophlus). Also, we remind that loss of triaenes can be are usually larger than the rest of the plesiasters. This is well ‘‘partial’’ if it takes place during the development (e.g. Alectona)so documented in Vulcanella (Annulastrella) [37,89] and Theneap increasing our knowledge in Astrophoridap larvae may shed some [100,101,102,103], so we suggest that the microxeas found in light on the classification and the evolution of triaenes. the Vulcanellidaep (and maybe later in the lithistids, Pachastrellap and Characella) may have originated from large plesiasters reduced Evolution of Microscleres in the Astrophoridap (Fig. 4) to two actines. Thrombidae species have a unique type of amphiaster with Sterrasters have been secondarily lost at least nine times p p p recurved spines at each end, not found anywhere else in the independently (p.0.95) (Fig. 4): in Penares , Erylus sp., Erylus p p Astrophoridap. It has been secondarily lost in some species of candidata, Melophlus sp., Geostelletta , Calthropella , E. megastylifera, R. Thrombus. It is unclear if their amphiasters are homologous to the globostelleta+Rhabdastrella sp. and R. intermedia. This clearly demon- more typical amphiasters observed in A. millari. Thrombidae also strates how common secondary loss of a microsclere is, even when have trichotriaenes, not found anywhere else in the Astrophoridap. it has a clear function (sterrasters form a strong barrier protecting Since trichotriaenes are fairly small (compared to true triaenes) the sponge). Interestingly, most of the secondary losses of and coexist with true triaenes in Yucatania; they may be derived sterrasters have occurred in shallow-water species, living in from a large microsclere, and are certainly not triaenes per se. tropical or temperate — never boreal or arctic — waters (Fig. 4). Seemingly, in Theneap and Vulcanella (Annulastrella) large plesiasters Actually, our results suggest that secondary loss of megascleres and have occasionally been considered as megascleres [81]. Tricho- microscleres are more common in shallow-water species. It is triaenes could therefore have originated from a form of plesiaster. therefore tempting to propose that secondary loss of spicules has The characteristic large diactines in Alectona are also thought to be been favored in tropical to temperate shallow-waters. This further derived from large asters [17]. Supporting this hypothesis are the suggests that environmental parameters such as lower pressure, large triactines found in some Alectona and the oxyasters found in higher water temperature and/or lower silica concentration could Thoosa. However, according to the position of A. millari in our be responsible for the loss of these sterrasters. Such parameters are tree, and if we are right about the reallocation of Thoosa with already known for their effect on spicule morphology [104, Alectona, these oxyasters are not homologous to the ones that 105,106,107], especially silica concentration that appears to have appeared later in the Ancorinidaep and the Geodiidaep. As for the played an important role in sponge evolution [108,109]. But since fusiform amphiasters found in Alectona, their origin remains there is insufficient evidence for our hypotheses, we refrain from unknown. Meanwhile, the diversified streptaster set (spirasters, further speculation along these lines. metasters, plesiasters) that developed in the Theneidae and Vulnellidaep may have been reduced to amphiasters in the ancestor Conclusion of Clade A. On one side, the Ancorinidaep share a close common This study is the first comprehensive molecular phylogenetic ancestor with the lithistids/Characella/Neamphius. On the other study of the Astrophorida. We obtained a well-resolved tree that side, the Geodiidaep share a close common ancestor with the newly suggested phylogenetic relationships between 89 species of defined Pachastrellidae. In both cases, we can hypothesize that a Astrophorida from nine families of sponges. Most incongruences shortening and disappearance of the shaft and/or compression of found between the current classification (Systema Porifera) and our amphiaster, spirasters or even sanidasters could have easily led to molecular tree systematically made sense in the light of the appearance of euasters. Indeed, such ‘intermediate’ forms of morphology (e.g. reallocated Ancorinidae, G. intuta, D. bucklandi, asters can be observed in Characella, Pachastrella [99], Dercitus [73] C. pachastrelloides), scattered biochemical data and homoplasic or Neophrissospongia [32]. Two independent appearances of processes (convergent evolution and secondary loss). The euasters in the Astrophoridap are not surprising in comparison taxonomic translation of this tree was a revision of the with their independent appearance in Thoosa, some Hadromerida Astrophorida for which we proposed new classifications: the and in Chondrilla (Chondrosida). The reversed evolution is also Linnaean classification includes all extant taxa belonging to the known: amphiasters are derived from euasters in the case of Erylus Astrophorida (File S2) while the phylogenetic classification amphiastera from Colombia (not sampled). According to our data, includes at the moment only clades supported by molecular data sterrasters have appeared once (p.0.65) in the ancestor of the and morphological data (File S1, Fig. 5). We propose in File S3 a Geodiidaep. Evolution of spherules seem to be possible from key to all the Astrophorida families, sub-families and genera microrhabds (as in Caminus [35]) or from asters (as in some incertae sedis. And Table S4 summarizes the nomenclatural Calthropella [73]). The sanidasters may have evolved from changes resulting from our study with respect to the name of amphiasters and/or microrhabds but our spicule reconstructions Astrophorida species. With addition of the eight families of do not support this at the moment (Fig. 4). We have nonetheless lithistids as well as the Thoosidae and Neamphius huxleyi, the observed sanidaster-like amphiasters (in Pachastrella abyssi) and Astrophorida became a larger order than previously considered, sanidaster-like microrhabds (in some Pachymatismap normani). We comprising ca 820 species [6]. However, the phylogenetic must stress that the intermediate nodes leading to the Ancorinidaep position of a few Astrophorida genera not sampled here is still and the Geodiidaep are poorly supported so these hypotheses need pending (File S2). The polyphyly of some genera (Ecionemia, to be tested with additional molecular markers. The origin of Rhabdastrella, Erylus, Stelletta) suggest that they should be tested on microrhabds is seemingly contentious. The limit between a species to species basis. Finally, other contentious groups need microxeas, sanidasters and microrhabds is ambiguous and to be tested as potential members of the Astrophoridap:somemay probably reflects their multiple appearances. They have inde- have been confused with aster-bearing Hadromerida (e.g. Jaspis pendently appeared in (some) Ecionemia, Pachastrellap,theErylinaep, vs. Hemiasterella) while others may have lost all their asters and

PLoS ONE | www.plosone.org 15 April 2011 | Volume 6 | Issue 4 | e18318 Astrophorida Phylogeny triaenes and are mixed in polyphyletic orders such as the Table S3 Morphological matrix of the Astrophorida species Halichondrida or Haplosclerida. sampled in this study. Our study is far from being the first study to show the potential (DOC) misleading nature of spicules and to question their utility in Table S4 Nomenclatural changes in the Linnaean and phylo- sponge taxonomy [22,110,111,112], especially with the numerous genetic classification as a result of our study. studies on the phenotypical plasticity of spicules (e.g. [113]) and (DOC) the recent outburst of cryptic species identification [114,115,116]. But this is certainly the first study to show how widespread File S1 Definition of new clades defined in this study (following convergent evolution and secondary loss can be in spicule the rules of the PhyloCode v.4c). evolution: they have taken place many times, in all taxa, in (DOC) megascleres and microscleres, even when these seem to be File S2 Proposal for a new Linnaean classification of the adaptative and under selective pressures. Our results show for the Astrophorida. first time the banality of spicule secondary loss (especially for (DOC) microscleres) and its potential as a synapomorphy (e.g. in File S3 Key to the Astrophorida families, sub-families and Geostellettap). With a sponge classification depending so much on genera incertae sedis. spicules, secondary loss of spicules should from now on be taken (DOC) more into account in future research on sponge taxonomy and phylogeny. Acknowledgments Supporting Information The authors wish to thank the shipboard parties and the crews of the R/V Hans Brattstro¨m (Marine Biological Station of the University of Bergen), the Figure S1 Molecular phylogeny of the Astrophorida obtained R/V Polarstern (ARK-XXII/1a), the R/V G. M. Dannevig (Marine Biological with maximum likelihood analyses (metREV+G model) of the Station of Flødevigen), the R/V Pourquoi Pas? (IFREMER), the R/V COI amino-acid dataset. Bootstrap values .50 are given at the Kommandor Jack (EMEPC 2007) and the R/V Almirante Gago Coutinho nodes (2,000 ML replicates). (EMEPC 2008). The authors wish to thank people that contributed to the sampling of this study: Bernard Picton and Claire Goodwin (National (TIF) Museums, Northern Ireland), Isabelle Domart-Coulon (Muse´um National Figure S2 Molecular phylogeny of the Astrophorida obtained d’Histoire Naturelle, Paris), Mary Kay Harper (University of Utah, Salt with maximum likelihood analyses (HKY+I+G model) of the COI Lake City) in collaboration with the NCDDG program and with G. Concepcion, Rob van Soest and Elly Beglinger (Zoo¨logisch Museum van nucleotide dataset. Bootstrap values .50 are given at the nodes de Universiteit van Amsterdam), Joseph R. Pawlik and Tse–Lynn Loh (2,000 ML replicates). (University of North Carolina, Wilmington), Scott Nichols (University of (TIF) California, USA), Marcel Jaspars (University of Aberdeen), IRD of Noume´a (New Caledonia), Shirley Sorokin (SARDI Aquatic Sciences, Figure S3 Molecular phylogeny of the Astrophorida obtained Adelaide) and Thierry Laperousaz (South Australian Museum, Adelaide), with maximum likelihood analyses (GTR+I+G model) of the 28S Thierry Perez (Centre d’Oce´anologie de Marseille, France), Kyle Sweeney (C1-D2) dataset. Bootstrap values .50 are given at the nodes and Grace McCormack (National University of Ireland, Galway, Ireland), (2,000 ML replicates). Magnus Tornes (Norway), Chris Proctor (Devon), Marina Cunha (TIF) (University of Aveira, Portugal) and Jeroen Ingels (University of Ghent). Geodia corticostylifera was collected by M. Klautau (permission number in Table S1 Locality of collection, museum voucher numbers and IBAMA: 1822255). We thank Nicole Boury-Esnault for valuable comments Genbank accession numbers for the sponge specimens used in this on an earlier draft of this paper. study. (DOC) Author Contributions Table S2 Sponge identification modifications after re-examina- Conceived and designed the experiments: PC HTR. Performed the tion of Astrophorida specimens from previous molecular phyloge- experiments: PC. Analyzed the data: PC. Contributed reagents/materials/ analysis tools: HTR CS JX JR. Wrote the paper: PC. Collection of netic and biochemistry studies. specimens: PC HTR CS JX JR. Identification of specimens: PC HTR JX. (DOC) Phylogenetic analyses: PC. Contributed to the paper: HTR CS JX JR.

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